Embryonic stem cells (ES cells) are stem cells established from an inner cell mass of mammalian blastocyst, and can be infinitely grown while maintaining their potential for differentiating into all types of cells (pluripotency). ES cell was first established in mouse in 1981, and gave rise to a milestone technique of gene function analysis using knockout mouse. Moreover, ever since establishment of human ES cell was reported in 1998, application to regenerative medicine has been highly expected. That is, functional recovery is designed by transplanting myocardial cells or nerve cells differentiated from the ES cell into patients with cardiac infarction or neurodegenerative disease.
While the cell transplantation therapy as represented by bone marrow transplantation in leukemia has already been put to practice, two problems of ensured supply of sufficient cells for transplantation and suppression of rejection. Using ES cell that divides semipermanently, the problem of ensured supply of sufficient cells can be solved altogether. Moreover, by combining the somatic cell clone technique, the rejection reaction can also be overcome. When ES cell is established from a clone embryo prepared from the somatic cell of the patient and used for transplantation, rejection cannot occur since the cell contains the same genes as those of the patient. Therefore, ES cell has a potential of solving both the two problems associated with the cell transplantation therapy.
Although ES cell has such a huge potential, since establishment and maintenance of human ES cell is difficult as compared to mouse ES cell, the development of certain establishment technique and culture technique is necessary. To establish a human ES cell, an embryo needs to be sacrificed. When combined with the somatic cell clone technique, it leads to the easy preparation of a human clone. To solve such an ethical problem, the development of a technique to directly produce a pluripotent ES-like cell from a somatic cell without using an embryo has been desired.
In such technique development, a gene specifically expressed in pluripotent cells such as ES cells ((ES cell associated transcript gene, hereinafter ECAT gene) plays an important role.
The ECAT gene becomes a marker to determine whether a cell is an ES cell. In addition, by combining the regulatory region that causes ES cell specific expression of ECAT gene and a drug resistant gene, an ES cell can be efficiently selected from a mixed culture of many kinds of cells (see patent reference 1). Moreover, by inducing expression of an ECAT gene in a somatic cell, conversion to an ES-like cell may be promoted.
A reported ECAT gene is the transcription factor Oct3 (also called Oct4 or POU5f1; hereinafter referred to as Oct3/4) gene. Although a similar gene has been reported in humans (hOct3/4 gene; see non-patent reference 1), there is no report of demonstrating the ES-cell-specific expression of the hOct-3/4 gene.
In recent years, in search of an unknown factor that positively maintains pluripotency in both ES cell and inner cell mass, our group has found nine genes specifically expressed in ES cells on the basis of the analysis of an EST database by Digital Differential Display, and designated them as ECAT1 gene-ECAT9 gene (see patent reference 1). Of these, ECAT4 is a factor also called Nanog, and has been shown to be an essential factor for the maintenance of the totipotency (pluripotency) of ES cells (see non-patent reference 2). ECAT5 is a factor also called ERas, and has been shown to promote the growth of ES cells (see non-patent reference 3).
In the aforementioned analysis, Oct3/4, UTF1, REX1 and the like reported to show experimental pluripotent cell-specific expression have additionally been identified, and therefore, the analysis has been established to be an extremely effective screening method.    patent reference 1: U.S. Pat. No. 6,146,888    patent reference 2: WO 02/097090    non-patent reference 1: Takeda et al., Nucleic Acids Research, 20: 4613-4620 (1992)    non-patent reference 2: Mitsui, K., et al., Cell, 113: 631-642 (2003)    non-patent reference 3: Takahashi, K., et al., Nature, 423: 541-545 (2003)